Essential hypertension induces serious complications such as cerebral stroke, ischemic heart disease, nephrosclerosis and the like, and these complications are basically related to angiopathy due to excessive growth of vascular smooth muscle cells (VSMC) and are targets of hypertension therapy. Further, after percutaneous transluminal coronary angioplasty (PTCA) for treatment of angina pectoris and myocardial infarction, restenosis occurs in about 40% of the cases, but there is no effective drug treatment therefor and is a big problem in the cardiovascular field. Histopathologically, it is known that TGF-β is involved in arterial proliferative diseases such as hypertensive vascular disease, neointimal formation after angioplasty and atherosclerosis. The cell growth in such diseases are thought to be suppressed by various mechanisms. One of the various mechanism is the inhibition of the transforming growth factor (TGF) expression.
TGF was discovered at first as a factor of mouse 3T3 cells transformed by Molony sarcoma virus (MSV) that converts normal cells to malignant cells, and is classified mainly to TGF-α and TGF-β. TGF-β is a part of the 112 amino acids at the C-terminal of the protein whose molecular weight is about 40,000 and which consists of 390-412 amino acids as a precursor and further forms a dimer (25 kDa) through a disulfide bond to have an activity.
TGF-β, which constitutes a protein family that controls growth and development of cells (non-patent document 1), is produced in various tissues such as blood vessels, platelets, liver, kidneys, heart muscle, lung, pancreas, skin, placenta, bone marrow and has a control activity on cell proliferation, extra-cellular matrix formation and immunity.
TGF-β suppresses proliferation of most of cells but has a 2 biphasic proliferating activity on mesenchymal cells such as fibroblast cells, vascular smooth muscle cells (VSMC) and the like. That is, in normal condition TGF-β suppresses the proliferation of these cells but stimulates the proliferation under the circumstances such as inflammation, mechanical stress and the like. These facts reveal that TGF-β is involved in neointimal formation after angiopathy by promoting the growth of VSMC and extra-cellular matrix formation. TGF-β is also involved in the formation of lesions of arteriosclerosis. Based on such information, it is believed that the local treatment of the blood vessel diseases aiming to control the effect of TGF-β could be effective to ease the arterial proliferative diseases described above.
Further, TGF-β is believed to be involved in the restenosis of renal artery after percutaneous renal angioplasty. These facts reveal that the specific TGF-β gene expression inhibitor of the present invention may be effective as a therapeutic drug for various proliferative vascular and stenotic diseases described above.
Further, astrocytes in the liver play an important role in the production of extracellular matrix in the process of fibril formation in the liver (non-patent document 2). The astrocytes are activated by TGF-β1, and the activated astrocytes induces TGF-β1 secretion from inflammatory cells in a damaged liver. At the same time the expression of the TGF-β1 receptor is enhanced in the activated astrocytes, and extracellular matrix proteins are increased by autocrine mechanism by TGF-β1 (non-patent document 3). These facts reveal that it is reasonable to assume the specific inhibitor of TGF-β1 gene expression of the present invention is effective as a therapeutic drug for various liver diseases described above.
Further, the expression of TGF-β is increased in parallel with the extracellular substrate in renal biopsy of model animals for nephropathies such as IgA nephropathy, focal glomeruloscerosis, crescentic nephritis, focal sclerotic lupus nephritis, diffuse proliferative lupus nephritis, diabetic nephropathy, hypertensive nephrosclerosis and the like and in renal biopsy tissue of patients of glomerulonephritis and diabetic nephropathy (non-patent document 4 and non-patent document 5). At the same time, Border et al. reported that administration of anti-TGF-β to Thy-1 nephritis rats inhibits accumulation of extracellular substrate in the renal glomeruli (non-patent document 5). These facts suggest that it is reasonable to assume the specific inhibitor of TGF-β1 gene expression of the present invention is effective as a therapeutic drug for various nephropathies described above.
Further, in the infarction focus at the scar forming stage in animal model of myocardial infarction, the expression of TGF-β is continuously elevated and involved in myocardial fibrillation (non-patent document 6). These facts suggest that it is reasonable to assume the specific inhibitor of TGF-β1 gene expression of the present invention is effective as a therapeutic drug for myocardial fibrillation after myocardial infarction.
Further, administration of anti-TGF-β antibodies and the soluble receptor of TGF-β to pulmonary fibrosis model animals improves pulmonary fibrosis (non-patent document 7). These facts suggest that it is reasonable to assume the specific inhibitor of TGF-β1 gene expression of the present invention is effective as a therapeutic drug for pulmonary fibrosis.
Further, there have been many reports on the high expression of TGF-β1 in human chronic pancreatitis, and administration of recombinant TGF-β to model animal of recurring acute pancreatitis induces fibril formation or high expression of the mRNA of fibronectin at the inflammation lesion of the pancreas. Conversely, it has been demonstrated that administration of a neutralizing antibody against TGF-β1, when pancreatitis model is prepared, inhibits the production of extracellular matrix and the expression of mRNA of I and III type collagen and fibronectine (non-patent document 8). These facts suggest that it is reasonable to assume the specific inhibitor of TGF-β1 gene expression of the present invention is effective as a therapeutic drug for fibril formation in chronic pancreatitis.
Further, it has been proposed that scleroderma may be caused by TGF-β, and Mori et al. reported that TGF-β induced fibrillation of the skin in model mice for dermal fibrillation (non-patent document 9). These facts suggest that it is reasonable to assume the specific inhibitor of TGF-β1 gene expression of the present invention is effective as a therapeutic drug for various skin fibrillation diseases.
Further, it has been reported that the expression of TGF-β mRNA is enhanced in megakaryocyte of bone marrow fibrosis patients (non-patent document 10), and the concentration of TGF-β in platelets takes a high value (non-patent document 11) and TGF-β concentration in plasma of the patients is significantly higher (non-patent document 12). According to Rameshwar et al., monocytes of bone marrow fibrosis patients activate, through adhesion, NF-k which induces IL-1 production, and IL-1 causes the bone marrow fibrosis by facilitating TGF-β production (non-patent document 13). These facts suggest that it is reasonable to assume the specific inhibitor of TGF-β1 gene expression of the present invention is effective as a therapeutic drug for bone marrow fibrosis.
Further, it has been reported that in the cell culture system of androgenetic alopecia patients with frontal hair loss, male hormone induces TGF-β1 from dermal papilla cells, and this TGF-β1 inhibits the growth of epidermal cells (non-patent document 14). These facts suggest that it is reasonable to assume the specific inhibitor of TGF-β1 gene expression of the present invention is effective as a therapeutic drug for androgenetic alopecia with frontal hair loss.
The method of inactivating a gene function in the reverse genetics has been used to analyze a specific gene function but also has a potential to be utilized in therapeutic application for virus infection, cancer and other diseases caused by abnormal gene expression. That is, it is known that the inactivation of the gene function can be achieved at DNA level by homologous recombination or at RNA level by antisense oligodeoxyribonucleotides and ribozymes. However, the methods using antisense oligonucleotides and ribozymes have problems that the target sequence is limited, the transfer of the antisense oligonucleotides and ribozymes to tissues and cells is inefficient, and they are prone to degradation by ribonuclease.
On the other hand, it has been reported that pyrrole-imidazole polyamides (hereinafter also referred to as Py-Im polyamide), unlike (deoxy)ribonucleotide reagents such as antisense reagents and ribozymes, can specifically recognize a DNA sequence and control the expression of a specific gene extracellularly.
Pyrrole-imidazole polyamides are a group of synthetic small molecules which are composed of N-methylpyrrole units (hereinafter also called Py) which is aromatic ring and N-methylimidazole units (hereinafter also called Im) (non-patent documents 15-17). Py and Im, continually coupled and folded, can assume a U-shaped conformation in the presence of γ-aminobutyrate. In the pyrrole-imidazole polyamides related to the present invention, N-methylpyrrole units (Py), N-methylimidazole units (Im) and γ-aminobutyrate units (also called γ-linker) are bound each other by amide bond (—C(═O)—NH—), and the general structure and production methods of the pyrrole-imidazole polyamides are publicly known (patent documents 1-3).
Synthetic polyamides can bind to a specific base pair in the minor groove of double helix DNA with a high affinity and specificity. The specific recognition of a base pair depends on the formation of a one-to-one pair between Py and Im. That is, in the U-shaped conformation in the minor groove of DNA, Py/Im pair targets a C-G base pair, Im/Py targets a G-C base pair, and Py/Py targets both an A-T base pair and a T-A base pair (non-patent documents 16-17). According to a recent study, it becomes clear that as the result of substituting a pyrrole ring of Py/Py pair with 3-hydroxypyrrole (Hp), the A-T condensation can be overcome by binding of Hp/Py preferentially to a T/A pair (non-patent document 18).
In general, the initiation of transcription is considered to be an important point of a gene control. For initiating transcription, several transcription factors are required to bind to specific recognition sequences in the promoter region of a gene. A polyamide in the minor groove may interfere with the gene control by blocking the binding of a transcription factor if the transcription factor plays an important role in the gene expression. This hypothesis has been proven to be correct in in vitro and in vivo experiments. An 8 membered ring Py-Im polyamide, which is bound inside the recognition site of zinc finger (the binding site of TFIIIA), inhibits the transcription of the 5S RNA gene (non-patent document 19). Polyamides that bind to a base pair sequence contiguous to a transcription factor sequence in a promoter of human immunodeficiency virus type 1 (HIV-1) blocks HIV-1 replication in human cells. These sequences include the TATA box, lymphocyte enhancer factor LEF-1 sequence and ETS-1 sequence (non-patent document 20). In contrast to these, a polyamide may also activate expression of a gene, by blocking repressor factor or replacing an original transcription factor (non-patent documents 21-23). UL122 mediated early protein 2 (IE86) of human cytomegalovirus (CMV) blocks the supply of RNA polymerase II to the promoter and inhibits the transcription of the corresponding genes (non-patent document 21). Synthetic polyamides can block the inhibition by IE86 and relieve the expression of the corresponding genes (non-patent document 22). The polyamide designed by Mapp acts as an artificial transcription factor and mediates the transcription reaction of the gene (non-patent document 23).
Patent document 1: Japanese Patent No. 3045706
Patent document 2: JP-A-2001-136974
Patent document 3: WO 03/000683 A1
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